TY - JOUR
T1 - Mitochondrial thioredoxin-responding off-on fluorescent probe
AU - Lee, Min Hee
AU - Han, Ji Hye
AU - Lee, Jae Hong
AU - Choi, Hyo Gil
AU - Kang, Chulhun
AU - Kim, Jong Seung
PY - 2012/10/17
Y1 - 2012/10/17
N2 - We synthesized a new probe, Mito-Naph, to visualize mitochondrial thioredoxin (Trx) activity in cells. A fluorescence off-on change is induced by disulfide cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular cyclization by the released thiolate to give a fluorescent product. By measuring the fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to 50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for Trx is (4.04 ± 0.26) × 103 (M s) -1, approximately 5000 times faster than that for GSH. The inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with other biological thiols containing amino acids in vitro and in vivo.
AB - We synthesized a new probe, Mito-Naph, to visualize mitochondrial thioredoxin (Trx) activity in cells. A fluorescence off-on change is induced by disulfide cleavage of the probe, resulting from a reaction with Trx and subsequent intramolecular cyclization by the released thiolate to give a fluorescent product. By measuring the fluorescence at 540 nm, Trx activity can be detected at nanomolar concentrations (down to 50 nM) well below its physiological levels. The in vitro and in vivo Trx preference of Mito-Naph was demonstrated by fluorometric and confocal microscopic experiments. In vitro kinetic analysis of the disulfide bond cleavage revealed that the second-order rate constant for Trx is (4.04 ± 0.26) × 103 (M s) -1, approximately 5000 times faster than that for GSH. The inhibition experiments involving PX-12, a selective inhibitor of Trx, also revealed that the emission from Mito-Naph significantly decreased in PX-12 dose-dependent manners, both in living cells and in cellular protein extracts. The Trx preference was further supported by an observation that the fluorescence intensity of rat liver extract was decreased according to the Trx depletion by immunoprecipitation. On the basis of these results, it is concluded that Mito-Naph preferentially reacts with Trx, compared with other biological thiols containing amino acids in vitro and in vivo.
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U2 - 10.1021/ja308446y
DO - 10.1021/ja308446y
M3 - Article
C2 - 23017013
AN - SCOPUS:84867547525
SN - 0002-7863
VL - 134
SP - 17314
EP - 17319
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 41
ER -